Genetic analysis has shown that there is a family of 5 unlinked MAL loci in yeast, any one of which is sufficient to convert maltose to glucose. The evidence obtained from our previous studies demonstrates that MAL1 and MAL6 are complex loci consisting of at least 3 genes: a maltose permease gene, a maltase gene and a regulatory gene whose product controls the coordinate induction of maltose permease and maltase. In addition, Southern blot hybridizations demonstrate that all 5 MAL loci share sequence homology. The main thrust of the proposal is to investigate in greater detail the organization, relatedness and the nature of the telomeric locations of the MAL loci using recombinant DNA and genetic techniques. We have now cloned MAL6, MAL2, MAL4 and two non-functional alleles of MAL1. Cosmid and Lambda vectors will be used to clone the remaining MAL loci. Cloned and subcloned MAL loci and adjacent sequences will be subjected to restriction enzyme, cross-hybridization and heteroduplex analyses to study their relatedness. Such analyses will also define the functional organization of the cluster of genes within the different MAL loci. In related studies, DNA sequencing of the MAL6 locus is well underway. The ubiquitous presence of the MAL1 locus (or one of its alleles) in many laboratory strains indicates that it may be the progenitor of the other MAL loci. Experiments will be directed to investigate how the MAL loci may have been dispersed. The mechanism of dispersion of the SUC loci encoding invertase may be similar to that of the MAL loci; SUC1 appears to have been transposed adjacent to mal1. The relationship of MAL1/SUC1, two very closely linked markers, will be investigated. An intensive genetic analysis will be continued on in vivo and in vitro generated Mal mutants, primarily at the MAL1 (and MAL6) loci in order to saturate the genetic map of these two loci. The genetic, physiological and physical analysis of these mutants should specifically allow us to: (1) Establish structure-function relationships; (2) provide appropriate recipients in complementation studies; (3) help to define the role of the regulatory protein and its site(s) of action; (4) examine genetic exchange between loci; and (5) help define the MALp and MALg functions designated by Naumov to be components of MAL loci. The genetic and physical analyses should allow us to determine the degree of functional and physical homology existing among all 5 MAL loci.